U.S. patent number 7,323,098 [Application Number 10/653,257] was granted by the patent office on 2008-01-29 for biosensor and measuring method using the same.
This patent grant is currently assigned to Matsushita Electric Industrial Co., Ltd.. Invention is credited to Mariko Miyashita, Toshihiko Yoshioka.
United States Patent |
7,323,098 |
Miyashita , et al. |
January 29, 2008 |
**Please see images for:
( Certificate of Correction ) ** |
Biosensor and measuring method using the same
Abstract
A biosensor capable of measuring the concentration of one or
more specific substances in one or more sample solutions almost
simultaneously is provided. The biosensor comprises a plurality of
sensor units, and each of the sensor units comprises an electrode
system including a working electrode and a counter electrode on an
insulating base plate and a reagent system including an
oxidoreductase and an electron mediator. The biosensor is so
configured that sample solutions supplied to the respective sensor
units reach the respective reagent systems at different times.
Specifically, each of the sensor units has a controlling system
between a sample supply inlet and the reagent system, and the
controlling system controls the time it takes for the sample
solution to reach the reagent system from the sample supply
inlet.
Inventors: |
Miyashita; Mariko (Nishinomiya,
JP), Yoshioka; Toshihiko (Hirakata, JP) |
Assignee: |
Matsushita Electric Industrial Co.,
Ltd. (Osaka, JP)
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Family
ID: |
31712282 |
Appl.
No.: |
10/653,257 |
Filed: |
September 3, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040040866 A1 |
Mar 4, 2004 |
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Foreign Application Priority Data
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Sep 3, 2002 [JP] |
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2002-257647 |
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Current U.S.
Class: |
205/777.5;
204/403.14; 204/411; 205/792 |
Current CPC
Class: |
C12Q
1/004 (20130101); G01N 27/3272 (20130101) |
Current International
Class: |
G01N
27/327 (20060101) |
Field of
Search: |
;204/403.01-403.15,409-412 ;205/777.5,778,792 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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4-284246 |
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Sep 1992 |
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JP |
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5-196596 |
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Aug 1993 |
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JP |
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10-267887 |
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Oct 1998 |
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JP |
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11-344460 |
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Dec 1999 |
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JP |
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WO 00/50630 |
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Aug 2000 |
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WO |
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WO 01/71328 |
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Sep 2001 |
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WO |
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Other References
JPO English language computer translation of Miyashita et al. (JP
11-344460 A) Dec. 14, 1999. cited by examiner .
CAPLUS abstract (JP 04-264246 A) Yoshioka et al. Sep. 21, 1992.
cited by examiner .
Chinese Office Action dated Jan. 26, 2007 (with English
Translation). cited by other.
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Primary Examiner: Noguerola; Alex
Attorney, Agent or Firm: McDermott Will & Emery LLP
Claims
The invention claimed is:
1. A biosensor comprising: a plurality of sensor units each
comprising an electrode system including a working electrode and a
counter electrode on an insulating base plate and a reagent system
including an oxidoreductase and an electron mediator on or near the
electrode system; a sample supply inlet provided for each of the
sensor units or for every some sensor units; and a plurality of
controlling systems for controlling the time it takes for a sample
solution to reach the reagent system from the sample supply inlet,
each controlling system being provided between the sample supply
inlet and each of the reagent systems of the sensor units, wherein
said respective controlling systems are different from one another
so that the sample solution supplied to the sample supply inlet
reaches the respective reagent systems of the sensor units at
different times; and wherein said sample supply inlet is provided
for each of the sensor units, and the biosensor has one working
electrode terminal connected in parallel to the respective working
electrodes of the sensor units and one counter electrode terminal
connected in parallel to the respective counter electrodes of the
sensor units.
2. A biosensor comprising: a plurality of sensor units each
comprising an electrode system including a working electrode and a
counter electrode on an insulating base plate and a reagent system
including an oxidoreductase and an electron mediator on or near the
electrode system; a sample supply inlet provided for each of the
sensor units or for every some sensor units; and a plurality of
controlling systems for controlling the time it takes for a sample
solution to reach the reagent system from the sample supply inlet,
each controlling system being provided between the sample supply
inlet and each of the reagent systems of the sensor units, wherein
said respective controlling systems are different from one another
so that the sample solution supplied to the sample supply inlet
reaches the respective reagent systems of the sensor units at
different times; and wherein said sample supply inlet is provided
for each of the sensor units, and each of the sensor units further
has a working electrode terminal connected to the working electrode
and a counter electrode terminal connected to the counter
electrode.
3. A biosensor comprising: a plurality of sensor units each
comprising an electrode system including a working electrode and a
counter electrode on an insulating base plate and a reagent system
including an oxidoreductase and an electron mediator on or near the
electrode system; a sample supply inlet provided for each of the
sensor units or for every some sensor units; and a plurality of
controlling systems for controlling the time it takes for a sample
solution to reach the reagent system from the sample supply inlet,
each controlling system being provided between the sample supply
inlet and each of the reagent systems of the sensor units, wherein
said respective controlling systems are different from one another
so that the sample solution supplied to the sample supply inlet
reaches the respective reagent systems of the sensor units at
different times; and wherein said controlling systems comprise a
layer comprising a hydrophilic polymer, and said respective
controlling systems of the senosr units are different from one
another in the speed with which the layer comprising a hydrophilic
polymer dissolves in the sample solution.
4. A biosensor comprising: a plurality of sensor units each
comprising an electrode system including a working electrode and a
counter electrode on an insulating base plate and a reagent system
including an oxidoreductase and an electron mediator on or near the
electrode system; a sample supply inlet provided for each of the
sensor units or for every some sensor units; and a plurality of
controlling systems for controlling the time it takes for a sample
solution to reach the reagent system from the sample supply inlet,
each controlling system being provided between the sample supply
inlet and each of the reagent systems of the sensor units, wherein
said respective controlling systems are different from one another
so that the sample solution supplied to the sample supply inlet
reaches the respective reagent systems of the sensor units at
different times; and wherein said controlling systems comprise a
layer of a porous material or a fibrous substance having pores
through which the sample solution passes, and said respective
controlling systems of the sensor units are different from one
another in the speed with which the sample solution passes through
the layer pf the porous material or the fibrous substance.
5. A method for determining the concentration of a specific
substance contained in each of sample solutions supplied to an
arbitrary number N of sensor units of the biosensor of claim 2,
which detects an electrochemical change of the electron mediator
caused by a reaction between the specific substance in the sample
solution and the oxidoreductase of the reagent system upon
dissolution of the reagent system into the supplied sample solution
after a lapse of a time period determined by said controlling
system, said electrochemical change being detected by measuring a
response current when a voltage is applied between the working
electrode and the counter electrode, said method comprising the
steps of: (a) supplying the sample solutions almost simultaneously
to the respective sample supply inlets of the sensor units; (b)
applying a voltage between the working electrode terminal and the
counter electrode terminal and measuring a response current N times
at intervals determined by the respective controlling systems of
the sensor units; and (c) determining the concentration of the
specific substance contained in each of the sample solutions
supplied to the respective sensor units from the N measured values
of the response current, wherein the sample solutions supplied to
the N sensor units are either the same sample solution or different
sample solutions.
6. A method for determining the concentration of a specific
substance contained in each of sample solutions supplied to an
arbitrary number N of sensor units of the biosensor of claim 3,
which detects an electrochemical change of the electron mediator
caused by a reaction between the specific substance in the sample
solution and the oxidoreductase of the reagent system upon
dissolution of the reagent system into the supplied sample solution
after a lapse of a time period determined by said controlling
system, said electrochemical change being detected by measuring a
response current when a voltage is applied between the working
electrode and the counter electrode, said method comprising the
steps of: (a) supplying the sample solutions almost simultaneously
to the respective sample supply inlets of the sensor units; (b)
applying a voltage between the working electrode terminal and the
counter electrode terminal and measuring a current response
sequentially unit by unit, a total of N times, at intervals
determined by the respective controlling systems of the sensor
units; and (c) determining the concentration of the specific
substance contained in each of the sample solutions supplied to the
respective sensor units from the N measured values of the response
current, wherein the sample solutions supplied to the N sensor
units are either the same sample solution or different sample
solutions.
7. A method for determining the concentration of a specific
substance contained in a sample solution supplied to an arbitrary
number N of sensor units of a biosensor comprising: a plurality of
sensor units each comprising an electrode system including a
working electrode and a counter electrode on an insulating base
plate and a reagent system including an oxidoreductase and an
electron mediator on or near the electrode system; a sample supply
inlet provided for each of the sensor units or for every some
sensor units; and a plurality of controlling systems for
controlling the time it takes for a sample solution to reach the
reagent system from the sample supply inlet, each controlling
system being provided between the sample supply inlet and each of
the reagent systems of the sensor units, wherein said sample supply
inlet is shared by the respective sensor units, and the biosensor
further has one working electrode terminal connected in parallel to
the respective working electrodes of the sensor units and one
counter electrode terminal connected in parallel to the respective
counter electrodes of the sensor units and; wherein said respective
controlling systems are different from one another so that the
sample solution supplied to the sample supply inlet reaches the
respective reagent systems of the sensor units at different times
wherein said sample supply inlet is shared by the respective sensor
units, and the biosensor further has one working electrode terminal
connected in parallel to the respective working electrodes of the
sensor units and one counter electrode terminal connected in
parallel to the respective counter electrodes of the sensor units;
said method comprising the steps of: (a) supplying the sample
solution to the sample supply inlet; (b) applying a voltage between
the working electrode terminal and the counter electrode terminal
and measuring a response current N times at intervals determined by
the respective controlling systems of the sensor units; and (c)
determining the concentration of the specific substance contained
in the sample solution supplied to the respective sensor units from
the N measured values of the response current, wherein the sample
solution supplied to the N sensor units contains one or more
specific substances, and the specific substances measured by the
respective sensor units are the same or different.
Description
BACKGROUND OF THE INVENTION
The present invention relates to a biosensor for measuring the
concentration of one or more specific substances contained in one
or more sample solutions and a measuring method using the
biosensor.
As a conventional biosensor for measuring the concentrations of a
plurality of specific substances simultaneously, there is a flow
injection type biosensor. One such example is Flow Injection
Analyzer (FIA) YSI MODEL 2700 SELECT (Yellow Spring Instrument Co.,
Inc.), which is a biosensor utilizing an immobilized enzyme
membrane and electrode reaction, and its operation for
simultaneously measuring glucose and L-lactic acid contained in a
sample solution is described below. First, the tip end of a sample
suction tube is immersed in a sample solution to suck the sample
solution into a measuring system, whereby the sample solution is
supplied into a sample chamber which is a measuring site. At the
same time, a certain amount of buffer solution is also introduced
into the sample chamber, where the sample solution and the buffer
solution are stirred and mixed with each other by a stirrer of the
sample chamber. The sample chamber is furnished with two electrodes
having an immobilized enzyme membrane of glucose oxidase
(hereinafter referred to as GOx) and an immobilized enzyme membrane
of lactate oxidase (hereinafter referred to as LOD), respectively.
After a lapse of a predetermined time period from the supply of the
sample solution and the buffer solution, an electrochemical
measurement is performed to calculate the concentrations of glucose
and L-lactic acid contained in the sample solution.
Also, an example of the biosensor for measuring a single specific
substance in a plurality of sample solutions successively is
Glucoroder GTX (A&T Co., Inc.), which measures the
concentration of glucose in blood, and its measuring operation is
described below. A certain amount of plasma, which is a sample
solution, is filled in the sample cups of a circular fraction
collector, and the sample cups are set to the fraction collector to
start a measurement. First, an aspirator automatically sucks a
certain amount of the sample solution from the first sample cup on
the fraction collector and moves to a measuring chamber containing
a buffer solution, where the sample solution is diluted with the
buffer solution by a predetermined dilution factor. Subsequently,
an immobilized enzyme electrode with an immobilized GOx membrane is
immersed in the sample solution in the measuring chamber, and after
a lapse of a predetermined time period, an electrochemical
measurement is performed. When the measurement of the first sample
cup is completed, the fraction collector turns so that the second
sample cup moves to the position of the first sample cup, and the
sample solution of the second sample cup is measured. In this way,
upon completion of the measurement of one sample cup, the fraction
collector turns so that another sample cup moves to the position
where the aspirator can operate, and measurements can be performed
successively.
Meanwhile, as a system for measuring a plurality of specific
substances in a sample solution without dilution or stirring with
ease, the following biosensor has been proposed in Japanese
Laid-Open Patent Publication No. Hei 5-196596. This biosensor has
electrode systems on opposite sides of the insulating base plate,
one on each side. On each of the electrode systems, a reaction
layer having a different enzyme or different combination of enzymes
is formed directly or indirectly. A method of measuring glucose and
fructose in a sample solution using this biosensor is described
below. First, an electrode system is formed by screen printing on
each side of the insulating base plate. A reaction layer containing
GOx is formed on the electrode system on one side of the base
plate, while a reaction layer containing fructose dehydrogenase
(hereinafter referred to as FDH) is formed on the electrode system
on the other side of the base plate. Further, a cover member is
joined to each side of the base plate to fabricate a sensor. The
cover member forms a sample solution supply pathway, through which
a sample solution is introduced into the electrode system, between
itself and the base plate. The enzyme reaction time of GOx is
shorter than that of FDH. Thus, by applying a voltage to the
electrode systems after one minute and two minutes, respectively,
from the simultaneous supply of the sample solution to the
respective sample supply inlets of the sensor, and measuring the
current five seconds later, the concentrations of glucose and
fructose in the sample solution can be quantified.
The above-described conventional biosensors of flow injection type
for measuring the concentrations of a plurality of specific
substances simultaneously need a buffer solution serving as a
carrier for making a measurement. In the above example, since the
buffer solutions of GOx and LOD are the same, the two components
can be measured simultaneously. However, if the appropriate pHs of
the enzymes for the plurality of specific substances to be measured
or the appropriate kinds of buffer solutions are different,
simultaneous measurement is impossible. Also, the measuring system
becomes large-scale, and the maintenance becomes complicated.
Also, the above-described conventional biosensor for measuring a
single specific substance contained in a plurality of sample
solutions needs a large-scale means, such as the fraction
collector, for supplying samples successively. Further, since
simultaneous measurement is not possible, the sample solutions
evaporate with passage of time, which may result in a decreased
measuring accuracy.
Further, the above-described biosensor having a plurality of
electrode systems on the base plate is unable to divide the current
of the plurality of electrode systems and detect the current of
each electrode system in the case where there is no difference in
enzyme reaction time among the enzymes and combination of enzymes
contained in a plurality of reaction layers. Thus, when there is no
difference in enzyme reaction time among the enzymes for a
plurality of specific substances to be measured and when the
specific substance to be measured is one, measurement is not
possible.
In view of these problems, an object of the present invention is to
provide a biosensor having a simple structure and capable of
measuring the concentrations of a plurality of specific substances
simultaneously even in the cases where the appropriate pHs of the
enzymes for the plurality of specific substances to be measured or
the appropriate kinds of buffer solutions are different and there
is no difference in enzyme reaction time among the enzymes.
Another object of the present invention is to provide a biosensor
having a simple structure and capable of measuring a single
specific substance contained in a plurality of sample solutions
continuously with high accuracy.
Further object of the present invention is to provide a measuring
method using the biosensor.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a biosensor comprising: a plurality
of sensor units each comprising an electrode system including a
working electrode and a counter electrode on an insulating base
plate and a reagent system including an oxidoreductase and an
electron mediator on or near the electrode system; a sample supply
inlet provided for each of the sensor units or for every some
sensor units; and a plurality of controlling systems for
controlling the time it takes for a sample solution to reach the
reagent system from the sample supply inlet, each controlling
system being provided between the sample supply inlet and each of
the reagent systems of the sensor units, wherein the respective
controlling systems are different from one another so that the
sample solution supplied to the sample supply inlet reaches the
respective reagent systems of the sensor units at different
times.
The biosensor of the present invention determines the concentration
of a specific substance contained in a sample solution supplied to
each of the sensor units in the following manner. When a sample
solution supplied to the sensor unit dissolves the reagent system
after a lapse of a time period determined by the controlling
system, the specific substance in the sample solution reacts with
the oxidoreductase of the reagent system to cause an
electrochemical change of the electron mediator, and this
electrochemical change is detected by measuring a response current
when a voltage is applied between the working electrode and the
counter electrode. This measuring principle is disclosed, for
example, in U.S. Pat. No. 5,120,420, which is incorporated herein
by reference in its entirety.
The biosensor of the present invention has the plurality of sensor
units, and has different configurations depending on whether one
sample supply inlet, from which a sample solution is supplied to
the electrode system, is shared by the respective sensor units or
is provided for each of the sensor units. Further, depending on
whether a measuring electrode terminal and a counter electrode
terminal are shared by the respective sensor units or are provided
for each of the sensor units in order to detect electrochemical
changes in the electrode systems of the sensor units, the biosensor
also has different configurations in terms of electric circuit, and
there are different measuring methods accordingly.
While the novel features of the invention are set forth
particularly in the appended claims, the invention, both as to
organization and content, will be better understood and
appreciated, along with other objects and features thereof, from
the following detailed description taken in conjunction with the
drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
FIG. 1 is a plane view of a biosensor in one embodiment of the
present invention from which the spacer and cover are omitted.
FIG. 2 is a decomposed perspective view of the biosensor of FIG. 1
from which the reagent system and controlling system are
omitted.
FIG. 3 is sectional views taken on line III--III of FIG. 1, showing
processes of forming an electrode system.
FIG. 4. is a sectional view of the biosensor with the reagent
system and controlling system taken on line III--III of FIG. 1.
FIG. 5 is a plane view of a biosensor in another embodiment of the
present invention from which the spacer and cover are omitted.
FIG. 6 is a decomposed perspective view of the biosensor of FIG. 5
from which the reagent system and controlling system are
omitted.
FIG. 7 is sectional views taken on line VII--VII of FIG. 5, showing
processes of forming an electrode system.
FIG. 8. is a sectional view of the biosensor with the reagent
system and controlling system taken on line VII--VII of FIG. 5.
FIG. 9 is a sectional view taken on line IX--IX of FIG. 5.
FIG. 10 is a sectional view of a sample solution supply pathway of
a biosensor in another embodiment of the present invention.
FIG. 11 is a perspective view of a cover of a biosensor in still
another embodiment of the present invention.
FIG. 12 is a sectional view taken on line XII--XII of FIG. 11.
FIG. 13 is a plane view of a biosensor in still another embodiment
of the present invention from which the spacer and cover are
omitted.
FIG. 14 is a decomposed perspective view of the biosensor of FIG.
13 from which the reagent system and controlling system are
omitted.
FIG. 15 is a decomposed perspective view of a biosensor in still
another embodiment of the present invention from which the reagent
system and controlling system are omitted.
DETAILED DESCRIPTION OF THE INVENTION
A biosensor of the present invention comprises a plurality of
sensor units, and each of the sensor units comprises: an electrode
system including a working electrode and a counter electrode; a
reagent system including an oxidoreductase and an electron mediator
on or near the electrode system; and a controlling system for
controlling the time it takes for a sample solution to reach the
reagent system from a sample supply inlet. The sample supply inlet
is provided for each of the sensor units or shared by the
respective sensor units, and the controlling system is provided
between the sample supply inlet and the reagent system. The
biosensor is so configured that the respective controlling systems
are different from one another so that the sample solution supplied
to the sample supply inlet reaches the respective reagent systems
of the sensor units at different times.
The reagent system is positioned such that at least part of the
reagent system comes in contact with the sample solution supplied
to the sensor unit. It is particularly preferable that the reagent
system be provided on or near the electrode system. The reagent
system may be provided in a state of being mixed with a conductive
material constituting the working electrode or the counter
electrode.
In a biosensor of a first preferable mode of the present invention,
each of the senor units has a sample supply inlet, and the
biosensor has one working electrode terminal connected in parallel
to the respective working electrodes of the sensor units and one
counter electrode terminal connected in parallel to the respective
counter electrodes of the sensor units.
A method for determining the concentration of a specific substance
contained in each of sample solutions supplied to an arbitrary
number N of sensor units of this biosensor comprises the steps of:
(a) supplying the sample solutions almost simultaneously to the
respective sample supply inlets of the sensor units; (b) applying a
voltage between the working electrode terminal and the counter
electrode terminal and measuring a response current N times at
intervals determined by the respective controlling systems of the
sensor units; and (c) determining the concentration of the specific
substance contained in each of the sample solutions supplied to the
respective sensor units from the N measured values of the response
current. In this case, the sample solutions supplied to the sensor
units may be the same sample solution or different sample
solutions.
In a biosensor of a second preferable mode of the present
invention, each of the sensor units has a sample supply inlet, and
each of the sensor units further has a working electrode terminal
connected to the working electrode and a counter electrode terminal
connected to the counter electrode.
A method for determining the concentration of a specific substance
contained in each of sample solutions supplied to an arbitrary
number N of sensor units of the second biosensor comprises the
steps of: (a) supplying the sample solutions almost simultaneously
to the respective sample supply inlets of the sensor units; (b)
applying a voltage between the working electrode terminal and the
counter electrode terminal and measuring a current response
sequentially unit by unit, a total of N times, at intervals
determined by the respective controlling systems of the sensor
units; and (c) determining the concentration of the specific
substance contained in each of the sample solutions supplied to the
respective sensor units from the N measured values of the response
current. The sample solutions supplied to the sensor units may be
the same sample solution or different sample solutions.
In a biosensor of a third preferable mode of the present invention,
the biosensor has one sample supply inlet shared by the respective
sensor units, and the biosensor further has one working electrode
terminal connected in parallel to the respective working electrodes
of the sensor units and one counter electrode terminal connected in
parallel to the respective counter electrodes of the sensor
units.
A method for determining the concentration of a specific substance
contained in a sample solution supplied to an arbitrary number N of
sensor units of the third biosensor comprises the steps of: (a)
supplying the sample solution to the sample supply inlet; (b)
applying a voltage between the working electrode terminal and the
counter electrode terminal and measuring a response current N times
at intervals determined by the respective controlling systems of
the sensor units; and (c) determining the concentration of the
specific substance contained in the sample solution supplied to the
respective sensor units from the N measured values of the response
current. The sample solution supplied to the N sensor units may
contain one or more specific substances, and the specific
substances measured by the respective sensor units may be the same
or different.
In a biosensor of a fourth preferable mode of the present
invention, the biosensor has one sample supply inlet shared by the
respective sensor units, and each of the sensor units has a working
electrode terminal connected to the working electrode and a counter
electrode terminal connected to the counter electrode.
A method for determining the concentration of a specific substance
contained in a sample solution supplied to an arbitrary number N of
sensor units of the fourth biosensor comprises the steps of: (a)
supplying the sample solution to the sample supply inlet; (b)
applying a voltage between the working electrode terminal and the
counter electrode terminal and measuring a current response
sequentially unit by unit, a total of N times, at intervals
determined by the respective controlling systems of the sensor
units; and (c) determining the concentration of the specific
substance contained in the sample solution supplied to the
respective sensor units from the N measured values of the response
current. The sample solution supplied to the N sensor units may
contain one or more specific substances, and the specific
substances measured by the respective sensor units may be the same
or different.
In the above-described measuring methods, it is preferable to
measure the response current of the electrode system of a sensor
unit after the working electrode and the counter electrode of the
sensor unit come in contact with a sample solution to cause liquid
junction. In other words, it is preferable to measure it after
detecting the change in electric resistance between the electrodes.
Since the liquid junction allows determination of the arrival of
the sample solution at the electrode system, accurate measurement
becomes possible.
A preferable controlling system which is used in the biosensor of
the present invention comprises a layer comprising a hydrophilic
polymer, and the respective controlling systems of the sensor units
are different from one another in the speed with which the layer
comprising a hydrophilic polymer dissolves in the sample
solution.
Another preferable controlling system comprises a layer of a porous
material or a fibrous substance having pores through which the
sample solution passes, and the respective controlling systems of
the sensor units are different from one another in the speed with
which the sample solution passes through the layer of the porous
material or the fibrous substance.
Still another preferable controlling system comprises a sample
solution supply pathway which extends from the sample supply inlet
to the reagent system, and the respective controlling systems are
different from one another in the length of the sample solution
supply pathway.
In the case of using the same hydrophilic polymer as the
controlling system for each of the sensor units, the speed with
which the layer comprising the hydrophilic polymer dissolves in the
sample solution may be changed from unit to unit by adjusting the
thickness, density or the like of the layer comprising the
hydrophilic polymer. Also, the use of hydrophilic polymers having
different solubilities allows the dissolution speeds to differ from
unit to unit.
Examples of the hydrophilic polymer which may be used in the
present invention include cellulose derivatives such as
carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl
cellulose, methyl cellulose, ethyl cellulose, and ethylhydroxyethyl
cellulose, polyamino acid such as polylysine, polystyrene sulfonic
acid, gelatin and its derivatives, polyacrylic acid and its salts,
polymethacrylic acid and its salts, starch and its derivatives, a
polymer of maleic anhydride or its salts, and agarose gel and its
derivatives.
The speed with which the sample solution passes through the layer
of the porous material or the fibrous substance having pores
serving as the controlling system may be adjusted by the thickness,
pore size, porosity or the like.
Examples of the porous material which may be used in the present
invention include porous glass, porous ceramics, porous metal such
as foamed metal and sintered metal, and activated carbon. Examples
of the layer of a fibrous substance include non-woven fabric, glass
fiber filter paper, and filter paper.
Embodiments of the present invention are specifically detailed
below with reference to drawings. The structural drawings used
herein are merely intended to facilitate understanding and are
therefore not necessarily accurate in terms of the relative size
and positional relation of the respective elements.
Embodiment 1
FIG. 1 is a plane view of a biosensor in Embodiment 1 from which
the spacer and cover are omitted. FIG. 2 is a decomposed
perspective view of the biosensor from which the reagent system and
controlling system are omitted. FIG. 3 contains sectional views
taken on line III--III of FIG. 1, showing processes of forming an
electrode system. FIG. 4 is a sectional view of the biosensor with
the reagent system and controlling system taken on line III--III of
FIG. 1.
A biosensor 10 of this embodiment is produced as follows. First, a
silver paste is printed on a base plate 2 made of an insulating
resin by screen printing to form a working electrode terminal 3 and
a working electrode lead 5 having 4 branches, as illustrated in
FIG. 3 (b). Next, a conductive carbon paste containing a resin
binder is printed to form 4 working electrodes 7, as illustrated in
FIG. 3 (c). Subsequently, an insulating paste is printed to form an
insulating layer 9, as illustrated in FIG. 3 (d). Further, a silver
paste is printed to form a counter electrode terminal 4 and a
counter electrode lead 6 having 4 branches, as illustrated in FIG.
3 (e). Lastly, a conductive carbon paste containing a resin binder
is printed to form 4 counter electrodes 8, as illustrated in FIG. 3
(f). The insulating layer 9 regulates the area of the working
electrodes 7 and insulates the working electrode lead 5 and the
working electrodes 7 connected thereto from the counter electrode
lead 6 and the counter electrodes 8 connected thereto.
Thereafter, a reagent system 11 including an enzyme and an electron
mediator is formed over the electrode systems each consisting of
the working electrode 7 and the counter electrode 8, and a
controlling system 17 serving as a means for controlling the time
it takes for a sample solution to reach the reagent system 11 is
formed over the respective reagent systems 11. The controlling
system 17 is, for example, a hydrophilic polymer layer, a porous
material or a layer of a fibrous substance.
Lastly, a spacer 12 having slits 15 and a cover 13 having air vents
16 are bonded in succession to the base plate 2 to complete the
biosensor 10. A sample solution supply pathway is formed in each of
the slits 15 of the spacer 12, and the open end of each of the
slits 15 serves as a sample supply inlet 14. When a sample solution
is brought in contact with the sample supply inlet 14, the sample
solution is introduced into the sample solution supply pathway by
capillarity to reach the reagent system 11 through the controlling
system 17.
In this way, the biosensor having four sensor units 1a, 1b, 1c and
1d is produced in this embodiment.
The principle on which the concentration of a specific substance in
a sample solution is measured using this biosensor is explained
below. The enzyme included in the reagent system 11 reacts with a
substrate (specific substance) contained in a sample solution to
reduce an oxidized form electron mediator. Upon application of a
voltage between the electrodes with the working electrode being
positive, a current oxidizing the reduced electron mediator flows
through the electrodes, and this current is measured. The measured
current value is proportional to the amount of the reduced electron
mediator resulting from the reaction between the enzyme and the
substrate, and the amount of the reduced electron mediator is
proportional to the concentration of the substrate contained in the
sample solution. Accordingly, the concentration of the substrate
contained in the sample solution can be calculated from the
obtained current value.
Next, the method of measuring the concentration of a specific
substance in a plurality of sample solutions using the biosensor of
this embodiment based on the above-described measuring principle is
explained in detail. The controlling system 17 controls the time it
takes for a sample solution to reach the reagent system 11 from the
sample supply inlet 14. The respective controlling systems 17 of
the sensor units are different from one another such that sample
solutions simultaneously supplied to the sample supply inlets reach
the respective reagent systems of the sensor units at different
times. Therefore, when sample solutions are supplied substantially
simultaneously to the respective sensor units of the biosensor, the
sample solutions reach the respective reagent systems 11 of the
sensor units at different times.
The measuring method using the biosensor of Embodiment 1 having an
arbitrary number N of sensor units is specifically described below.
Time measurement is started when sample solutions are
simultaneously supplied to the respective sample supply inlets of
the sensor units. One of the sample solutions reaches the reagent
system of one of the sensor units (first sensor unit) fastest to
cause a reaction at its electrode system. The current resulting
from this reaction and flowing between the measuring terminals 3
and 4 at time t.sub.1, is taken as I.sub.t1. Since the sample
solutions supplied to the other sensor units than the first sensor
unit have not reached their reagent systems then, the current
I.sub.1(t1) flowing through the electrode system of the first
sensor unit at time t.sub.1, is derived as follows:
I.sub.1(.sub.t1)=I.sub.t1 The calibration curb showing the relation
between the substrate concentration and the current value in the
first sensor unit at time t.sub.1 is expressed by a function
.alpha..sub.1(.sub.t1)(i) wherein i is a current value. Then, the
concentration C.sub.1 of the specific substance in the sample
solution measured based on the reaction at the electrode system of
the first sensor unit is derived as follows:
C.sub.1=.alpha..sub.1(t1){I.sub.1(t1)}
Next, another reaction takes place at the electrode system when
another sample solution reaches the reagent system of the second
sensor unit the second fastest. The current resulting from the
reactions at the electrode systems of the first and second sensor
units and flowing between the measuring terminals 3 and 4 at time
t.sub.2 is taken as I.sub.t2. Since the sample solutions supplied
to the third and subsequent sensor units have not reached their
reagent systems then, it is only the electrode systems of the first
and second sensor units that the current is flowing through at time
t.sub.2. By taking the current flowing through the electrode system
of the first sensor unit at time t.sub.2 as I.sub.1(t2) and the
current flowing through the electrode system of the second sensor
unit at time t.sub.2 as I.sub.2(.sub.t2), the current I.sub.t2
flowing through the whole biosensor is derived as follows:
I.sub.t2=I.sub.1(t2)+I.sub.2(t2)
Meanwhile, the concentration C.sub.1 at time t.sub.1 has been
already obtained, and the calibration curb showing the relation
between the substrate concentration and the current value in the
first sensor unit at time t.sub.2 is expressed by a function
.alpha..sub.1(t2)(i). Then, the concentration C.sub.1 is derived as
follows: C.sub.1=.alpha..sub.1(t2){I.sub.1(t2)} Therefore,
I.sub.1(t2)=.alpha..sub.1(t2).sup.-1(C.sub.1), and
I.sub.2(t2)=I.sub.t2-I.sub.1(t2)=l.sub.t2-.alpha..sub.1(t2).sup.-1(C.sub.-
1)
Meanwhile, the calibration curb showing the relation between the
substrate concentration and the current value in the second sensor
unit at time t.sub.2 is expressed by a function
.alpha..sub.2(t2)(i). Then, the concentration C.sub.2 is derived as
follows:
C.sub.2=.alpha..sub.2(t2){I.sub.t2-.alpha..sub.1(t2).sup.-1(C.sub.1)}
In this way, the sample solutions sequentially reach the reagent
systems of the sensor units, and the current resulting from the
reaction at the electrode system to which the sample solution has
newly reached adds to the current flowing between the terminals 3
and 4. Accordingly, the substrate concentration C.sub.n obtained
from the current resulting from the reaction at the electrode
system of the Nth sensor unit can be expressed as follows:
C.sub.n=.alpha..sub.n(tn)[I.sub.tn-{.alpha..sub.1(tn).sup.-1(C.sub.1+.
. . +.alpha..sub.n-1(tn).sup.-1(C.sub.n-1)}
Since the biosensor of this embodiment comprises the plurality of
sensor units each having the reagent system, the enzyme and buffer
contained in the reagent system of each of the sensor units can be
set appropriately depending on the substance to be measured. Thus,
even in the case where the appropriate pHs of the enzymes for a
plurality of specific substances to be measured or the appropriate
kinds of buffers are different, the concentrations of the plurality
of specific substances can be obtained by a series of operations of
measuring the current flowing between the measuring terminals 3 and
4 at intervals over time. The biosensor of this embodiment is
particularly effective when there is no difference in enzyme
reaction time among the enzymes of the respective sensor units.
Also, the biosensor of this embodiment is so configured that the
sample solutions are retained in the sample solution supply
pathways. Thus, the measurement is not affected by the evaporation
of the sample solutions although it takes different times for the
sample solutions to reach the electrode systems. Therefore, it is
possible to measure the concentration of a single specific
substance contained in a plurality of sample solutions with high
accuracy.
The biosensor of this embodiment needs only one pair of terminals
for detecting the current resulting from the reactions at the
electrode systems of the respective sensor units, so the number of
sensor units can be increased or decreased freely. This makes it
possible to freely change the kind of the specific substance and
the number of samples to be measured by the series of operations.
Also, since only one pair of terminals is used, only one pair of
connectors is necessary for mounting the sensor onto a measuring
device, so that it is possible to simplify the structure of the
measuring device and downsize the measuring device.
Embodiment 2
FIG. 5 is a plane view of a biosensor in Embodiment 2 from which
the spacer and cover are omitted. FIG. 6 is a decomposed
perspective view of the biosensor from which the reagent system and
controlling system are omitted. FIG. 7 contains sectional views
taken on line VII--VII of FIG. 5, showing processes of forming an
electrode system. FIG. 8 is a sectional view of the biosensor with
the reagent system and controlling system taken on line VII--VII of
FIG. 5.
A biosensor 20 of this embodiment is produced as follows. First, a
silver paste is printed on a base plate 22 made of an insulating
resin by screen printing to form 4 working electrode terminals 23,
4 counter electrode terminals 24, 4 working electrode leads 25, and
4 counter electrode leads 26, as illustrated in FIG. 7 (a). Next, a
conductive carbon paste containing a resin binder is printed to
form 4 working electrodes 27, as illustrated in FIG. 7 (b).
Subsequently, an insulating paste is printed to form an insulating
layer 29, as illustrated in FIG. 7 (c). Lastly, a conductive carbon
paste containing a resin binder is printed to form 4 counter
electrodes 28, as illustrated in FIG. 7 (d).
Thereafter, a reagent system 31 including an enzyme and an electron
mediator is formed over the respective electrode systems, and a
controlling system 37 serving as a means for controlling the time
it takes for a sample solution to reach the reagent system is
formed over the respective reagent systems 31. The controlling
system 37 is, for example, a hydrophilic polymer layer, a porous
material or a layer of a fibrous substance.
Lastly, a spacer 32 having slits 35 and a cover 33 having air vents
36 are bonded in succession to the base plate 22 to complete the
biosensor 20. A sample solution supply pathway is formed in each of
the slits 35 of the spacer 32, and the open end of each of the
slits 35 serves as a sample supply inlet 34. When a sample solution
is brought in contact with the sample supply inlet 34, the sample
solution is introduced into the sample solution supply pathway by
capillarity to reach the reagent system 31.
FIG. 9 is a sectional view taken on line IX--IX of FIG. 5. The
controlling system 37 is a hydrophilic polymer layer in this case.
The sample solution supplied to the sample supply inlet 34 flows
through the sample solution supply pathway formed in the slit 35
toward the air vent 36 by capillarity. When the sample solution
dissolves the hydrophilic polymer layer of the controlling system,
it then dissolves the reagent system 31 to initiate an enzyme
reaction.
FIG. 10 illustrates an example of a controlling system 47 being
ceramics or a layer of a fibrous substance. In the sample solution
supply pathway, the controlling system 47 is provided between the
sample supply inlet and the reagent system 31, and a sample
solution which has passed through the controlling system 47 reaches
and dissolves the reagent system 31.
FIG. 11 illustrates a cover 42 that is used instead of the spacer
32 and the cover 33 of FIG. 6. FIG. 12 is a sectional view of a
biosensor using the cover 42 taken on line XII--XII of FIG. 11. The
cover 42 has through holes 44 serving as sample supply inlets. A
sample solution supplied to each of the through holes 44 comes in
contact with the controlling system 37 and dissolves or permeates
through the controlling system 37 to reach the reagent system
31.
It is needless to say that the structures as illustrated in FIGS. 9
to 12 are also applicable to Embodiment 1.
In this way, the biosensor having four sensor units 21a, 21b, 21c
and 21d is produced in this embodiment.
The biosensor of this embodiment is different from that of
Embodiment 1 in that each of the sensor units has terminals for
measuring current, but its measuring principle of calculating the
concentration of a substrate in a sample solution from the current
flowing through the electrode system is the same as that of the
biosensor of Embodiment 1. That is, a voltage is sequentially
applied between the terminals of the electrodes of the sensor units
unit by unit at intervals determined by the differences in
structure of the respective controlling systems, and from the
current values obtained upon the voltage applications, the
concentration of the substrate contained in each of the sample
solutions supplied to the respective sensor units can be
calculated. Since the values measured by each of the sensor units
are not affected by other sensor units, more accurate measurement
becomes possible.
Embodiment 3
FIG. 13 is a plane view of a biosensor in Embodiment 3 from which
the spacer and cover are omitted. FIG. 14 is a decomposed
perspective view of the biosensor from which the reagent system and
controlling system are omitted.
A biosensor 50 of this embodiment is produced as follows. In the
same manner as in Embodiment 1, a working electrode terminal 53, a
working electrode lead 55 having 4 branches, 4 working electrodes
57, an insulating layer 59, a counter electrode terminal 54, a
counter electrode lead having 4 branches 56, and 4 counter
electrodes 58 are formed on a base plate 52. Subsequently, a
reagent system is formed over the respective electrode systems. A
spacer 62 of this embodiment has slits 65a, 65b, 65c , and 65d
having different lengths for forming respective sample solution
supply pathways of sensor units, and these slits communicate with
one opening 64 which serves as a sample supply inlet. The spacer 62
and a cover 63 having four air vents 66 are bonded to the base
plate 52 to complete the biosensor. The biosensor of this
embodiment is so configured that a sample solution supplied to the
one sample supply inlet is supplied to the respective sensor units
through the different-length sample solution supply pathways.
Because of the difference in length of the sample solution supply
pathways, the sample solution supplied to the sample supply inlet
reaches the respective reagent systems of the sensor units at
different times. In the case where there is not sufficient
difference in arrival time of the sample solutions at the reagent
systems of the sensor units, the biosensor may further comprise a
controlling systems as described in Embodiment 1 or 2. Although
this embodiment uses a pair of terminals (a working electrode
terminal and a counter electrode terminal) which is shared by the
respective sensor units, a pair of terminals may be provided for
each of the sensor units in the same manner as in Embodiment 2.
The biosensor of this embodiment is suited for measurement of
different specific substances in a sample solution.
In Embodiments 1 and 2, one sample supply inlet was provided for
each of the sensor units, but one sample supply inlet may be shared
by the respective sensor units in the same manner as in Embodiment
3. In this case, the sample solution supply pathways extending from
the one sample supply inlet to the respective reagent systems of
the sensor units may have the same length or different lengths.
In Embodiment 3, a pair of measuring terminals was used, but a pair
of measuring terminals may be provided for each of the sensor units
in the same manner as in Embodiment 2. In Embodiment 3, one sample
supply inlet was shared by the respective sensor units, but one
sample supply inlet may be provided for each of the sensor units as
illustrated in FIG. 15. In FIG. 15, instead of the spacer 62 of
FIG. 14 having one sample supply inlet, a spacer 72 having slits
75a, 75b, 75c and 75d of different lengths for forming sample
solution supply pathways and 4 sample supply inlets 74 for the
respective sensor units is used.
The measuring methods using the biosensors of the present invention
are effective when a sample solution containing a plurality of
substrates is supplied to the respective sensor units and the
sensor units measure different substrates. These measuring methods
are also effective when different sample solutions containing one
or more substrates are supplied to the respective sensor units and
the sensor units measure the same substrate or different
substrates.
The present invention is more specifically detailed below by way of
concrete examples.
EXAMPLE 1
A method of measuring glucose, fructose, L-lactic acid and alcohol
contained in sample solutions using the biosensor of FIG. 1 is
described. The reagent system 11 of each of the sensor units
contains the following on the electrode system. Sensor unit la for
measuring glucose Enzyme: GOx Electron mediator: potassium
ferricyanide Sensor unit lb for measuring fructose Enzyme: FDH
Electron mediator: potassium ferricyanide Sensor unit lc for
measuring L-lactic acid Enzyme: LOD Electron mediator: potassium
ferricyanide Sensor unit ld for measuring alcohol Enzyme: alcohol
dehydrogenase (ADH), diaphorase Coenzyme: .beta.-nicotinamide
adenin dinucleotide Electron mediator: potassium ferricyanide
Ethanol solutions having polyvinyl pyrrolidone (hereinafter
referred to as PVP) concentrations of 0, 0.5, 1.0, and 1.5% are
dropped over the reagent systems 11 on the electrode systems of the
sensor units 1a, 1b, 1c and 1d, respectively, and are dried to form
PVP layers. The PVP layers serve as the controlling systems 17 for
controlling the permeation of the sample solutions. The thicknesses
of the respective PVP layers of the sensor units are different from
one another. Due to this difference in PVP layer thickness, even
when sample solutions are simultaneously supplied to the respective
sensor units, the sample solutions reach the respective reagent
systems 11 at intervals of approximately 30 seconds. That is, the
sample solution supplied to the sensor unit la reaches the reagent
system first, and the sample solutions supplied to the sensor units
1b, 1c and 1d sequentially reach the reagent systems with a time
lag of approximately 30 seconds.
Sample solutions are supplied almost simultaneously to the
respective sample supply inlets of the sensor units of the
biosensor as described above. Since the sensor unit 1a for
measuring glucose has no PVP layer over the reagent system, the
sample solution which has reached the reagent system immediately
dissolves the reagent system over the electrode system. Upon the
dissolution, the working electrode 7 and the counter electrode 8
are electrically connected by liquid junction to cause a change in
impedance between the electrodes. By this impedance change, the
arrival of the sample solution at the electrode system is detected,
and simultaneously with this detection, time measurement is
started. After a lapse of 25 seconds, a voltage is applied between
the electrodes with the working electrode being positive, and 5
seconds later, the current flowing between the electrodes is
measured. From the current value measured, glucose concentration is
calculated.
Almost simultaneously with the measurement of the current, the PVP
layer of the sensor unit 1b for measuring fructose dissolves in the
sample solution, followed by the dissolution of the reagent system
11. Fifty five seconds after the liquid junction of the electrode
system of the sensor unit 1a, a voltage is applied between the
electrodes of the sensor unit 1b, and 5 seconds later, the current
is measured. The measured current value is the sum of the current
value derived from the reaction of fructose and FDH and the current
value derived from the reaction at the electrode system of the
sensor unit 1a to which the sample solution has already reached. By
subtracting the current value corresponding to the already obtained
glucose concentration from this measured current value, the current
value derived from the oxidation reaction of fructose can be
obtained. Accordingly, fructose concentration can be
calculated.
In this way, the concentrations of L-lactic acid and alcohol can
also be obtained.
EXAMPLE 2
A method of simultaneously measuring glucose contained in four
different sample solutions using the biosensor of FIG. 5 is
described. The reagent system 31 containing the enzyme GOx and the
electron mediator potassium ferricyanide is provided over the
respective electrode systems of the sensor units. Subsequently, a
glass fiber filter paper having a different thickness and a shape
substantially the same as the electrode system is provided as the
controlling system 37 over the respective reagent systems 31. When
sample solutions are supplied to the respective sample supply
inlets of the sensor units of the biosensor as described above, the
sample solutions reach the reagent systems 31 of the sensor units
at different times depending on the thickness of the glass fiber
filter paper. By utilizing this difference in arrival time among
the four sample solutions, a voltage is sequentially applied
between the terminals of the respective sensor units at
predetermined intervals, and the current which oxidizes the reduced
electron mediator is measured. In this way, it is possible to
calculate glucose concentrations in the four different sample
solutions.
In Examples 1 and 2, PVP (hydrophilic polymer) and glass fiber
filter paper (fibrous substance) were used as the means for
controlling the time it takes for the sample solution to arrive at
the reagent system 11 or 31. However, this is not to be construed
as limiting the present invention.
As described above, even in the case where the appropriate pHs of
the enzymes for a plurality of specific substances to be measured
or the appropriate kinds of buffers are different, or in the case
where there is no difference in reaction time among the enzymes,
the present invention makes it possible to determine the
concentrations of the plurality of specific substances by a series
of operations with ease and high accuracy. The present invention
also makes it possible to measure a single specific substance
contained in a plurality of sample solutions in a continuous manner
by a series of operations.
Although the present invention has been described in terms of the
presently preferred embodiments, it is to be understood that such
disclosure is not to be interpreted as limiting. Various
alterations and modifications will no doubt become apparent to
those skilled in the art to which the present invention pertains,
after having read the above disclosure. Accordingly, it is intended
that the appended claims be interpreted as covering all alterations
and modifications as fall within the true spirit and scope of the
invention.
* * * * *